agrawal and konno 2009 latex annual review

ANRV393-ES40-15 ARI 1 October 2009 11:56

Latex: A Model forUnderstanding Mechanisms,Ecology, and Evolution of PlantDefense Against HerbivoryAnurag A. Agrawal1 and Kotaro Konno2

1Department of Ecology and Evolutionary Biology, Department of Entomology, and CornellCenter for a Sustainable Future, Cornell University, Ithaca, New York 14853-2701;email: [email protected] Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8634, Japan;email: [email protected]

Annu. Rev. Ecol. Evol. Syst. 2009. 40:311–31

First published online as a Review in Advance onAugust 31, 2009

The Annual Review of Ecology, Evolution, andSystematics is online at ecolsys.annualreviews.org

This article’s doi:10.1146/annurev.ecolsys.110308.120307

Copyright c© 2009 by Annual Reviews.All rights reserved

1543-592X/09/1201-0311$20.00

Key Words

chemical ecology, coevolution, insect behavior, laticifer, plant-insectinteractions, plant resistance

AbstractLatex is a sticky emulsion that exudes upon damage from specialized canals inabout 10% of flowering plant species. Latex has no known primary metabolicfunction and has been strongly implicated in defense against herbivorous in-sects. Here we review historical hypotheses about the function of latex, evi-dence that it serves as a potent defense, and the chemistry and mode of actionof the major constituent defense chemicals and proteins across a diversity ofplant species. We further attempt to synthesize the characteristics of latex asa coordinated plant defense system. Herbivores that feed on latex-bearingplants typically evade contact with latex by severing the laticifers or feedingintercellularly, or may possess physiological adaptations. Convergent evolu-tion appears to be rampant both in plants with latex and insects that exploitlatex-bearing plants. Because latex shows phenotypic plasticity, heritability,and macoevolutionary lability, it is an ideal system to study plant-herbivoreinteractions using evolutionary approaches.

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Mandibulate: refersto insects that havemandibles for biting orchewing leaf tissue (asopposed to having aproboscis or stylet forsucking)

Latex: a milkysuspension oremulsion of particlesin an aqueous fluid,usually held underpressure in living plantcells (lacticifers)

Hevea brasiliensis orPara rubber tree(Euphorbiaceae):native to the Amazon,and perhaps thebest-studied latex-producing speciesbecause of theeconomic importanceof rubber

Asclepias: a genus ofherbaceous perennialplants with ≈140species in the Americasand named for theirexuding milky latex,commonly calledmilkweeds

INTRODUCTION

About 10% of all flowering plant species (angiosperms) exude latex upon tissue damage andthis latex has no known function in primary metabolism (in terms of plant resource acquisitionand allocation) (Farrell et al. 1991, Hunter 1994, Lewinsohn 1991, Metcalf 1967). Over thepast 20 years, a growing literature has emerged on latex, its biochemistry, and its ecologicaland evolutionary consequences. Both circumstantial and experimental evidence points to latexas a potent plant defense against mandibulate herbivores. Here we review various aspects ofthe chemical ecology and evolutionary biology of latex with special reference to plant-herbivoreinteractions. We use latex as a model to address general conceptually motivated questions thatscientists might ask about any plant defense (Sidebar: A set of conceptually motivated questionsaddressing the mechanisms, ecology, and evolution of any plant defense trait).

Not unlike the blood of animals, when the tissues of latex-bearing plants are damaged, latexoozes out, becomes sticky when exposed to air, and quickly coagulates. Sometimes latex exudationcan be remarkably abundant, like a squirt of toxic white glue, whereas at other times it maybe difficult to detect because it is clear or barely exudes (Agrawal et al. 2008, Metcalf 1967,Shukla & Krishna Murti 1971). Latex is sometimes colored yellow, orange, or red, such as thatof Cannabis (Cannabaceae). Latex is well known for its sticky properties, which have been used toproduce rubber (from Hevea brasiliensis Euphorbiaceae and other species), chicle from Manilkaraspp. (Sapotaceae) used in chewing gum, and lacquers from phenols in the latex of plants in theAnacardiaceae. Latex from various plant species contains bioactive compounds including alkaloidssuch as morphine in Papaver spp. (Papaveraceae); cardiac glycosides in Asclepias spp. (Apocynaceae);terpenes such as the sesquiterpene lactone, lactucin, from lettuce (Lactuca spp. Asteraceae); anddigestive cysteine proteases in Carica papaya (Caricaceae) and Ficus spp. (Moraceae) (see sectionon Biochemistry and Mode of Action).

As evidenced from the above list of plant families with latex-bearing species, latex is extraordi-narily common. Among flowering plants, over 20,000 species (from over 40 families in multiplelineages) contain latex (Farrell et al. 1991, Hunter 1994, Lewinsohn 1991, Metcalf 1967). Latex isfound in dicotyledonous and monocotyledonous (e.g., Liliaceae) plants. This finding, that nearly10% of families and species produce latex, implies that latex is a highly convergent trait (that is,

A SET OF CONCEPTUALLY MOTIVATED QUESTIONS ADDRESSING THEMECHANISMS, ECOLOGY, AND EVOLUTION OF ANY PLANT DEFENSE TRAIT

What is the mode of action? Are the constituents that make up the defense redundant, additive, or synergistic?Is this defense tied into primary plant metabolism, protection from abiotic stress, energy storage, or waste? Howspecific are the effects of the defense against a diversity of attackers? Does this defense interact with predation?How do resources and abiotic environment modulate expression of the trait? Are there ontogenetic shifts in theexpression of this trait? Is the defense inducible by herbivory? Which plant hormones regulate defense investment?Is there specificity in the elicitation of the induced response? How do herbivores cope with this defense? In whatsorts of communities is this defense present, and what are the consequences of herbivory at the community level?Is there heritable variation for the expression of the defense? What maintains this heritable variation (allocation orecological costs, spatial or temporal variation in the benefits)? How does this defense trait covary with other defensetraits within the species? Do herbivores impose natural selection on this trait? How evolutionarily conserved is thedefense in a clade of related plants? Is the defense adapted to particular populations or habitat types? Does thisdefense show phylogenetic patterns or trends? Any evidence for this defense as a key innovation?

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Resin: plant exudatescommon in conifersand some angiosperms(e.g., Anacardiaceae),rich in terpenoids andphenolics, and storedin intercelluar spaces

Mucilage: plant-produced stickypolysaccharides thatare often clear andexude from the phloemfollowing damage(common in cucurbits)

Gum: distinct fromlatex, resin, ormucilage; these watersolublepolysaccharides exudefrom cellular cavitiesor bark (as inRosaceous fruit trees)

Laticifer: anelongated cell with twomain morphologicaltypes (articulated ornonarticulated)produced in any plantpart, serving totransport latex

David Dussourd:American biologist atthe University ofCentral Arkansas whohas been the leadingscientist unraveling themechanisms of latex asa plant defense

has evolved independently multiple times) and that latex is likely encountered by many herbivorespecies. In addition, latex has been reported in mushrooms (e.g., Lactarius spp.), conifers (e.g., Gne-tum spp.) and pteridophytes (Metcalf 1967). Both in terms of absolute and proportional estimates,tropical plant families (and species overall) are more likely to produce latex than are temperategroups (Lewinsohn 1991). Indeed, some 14% of tropical plant species produced latex compared to6% of temperate species, and this distribution is not independent of plant phylogeny (Lewinsohn1991). Latex is phylogenetically conserved in the above-mentioned plant families, and these andother latex-bearing families may be overrepresented in the tropics.

ANATOMY

Latex can be distinguished from resins, mucilages, and gums, which also exude upon tissue damage.Resins are typically composed of terpenoids and phenolics and delivered from intercellular spaces,sometimes called ducts (not from living cells like laticifers) (Langenheim 2003). For example,Bursera schlechtendalii stores toxic terpenes in pressurized resin ducts, which, following damage,can squirt over 1 m and may drench an herbivore and coat the leaf surface (Becerra 1994). Althoughthe phloem primarily transports products of photosynthesis, in some plants such as Cucumis spp.(Cucurbitaceae), the pressurized phloem sap exudes upon damage and, like latex, gels into a stickysubstance. Indeed, the sap of cucurbits additionally contains various plant defense compounds(Dussourd 1997, Dussourd & Denno 1991, McCloud et al. 1995). Latex tends to be more phy-tochemically diverse than resins, mucilages, and gums, and often contains complex mixtures ofterpenoids, phenolics, proteins, and alkaloids (Langenheim 2003).

Laticifers take two main morphological forms (Figure 1). Nonarticulating laticifers are formedfrom single cells that often branch, but do not loop or reconnect (Dussourd & Denno 1991, Pickard2008). This form is typified by the laticifers of the milkweeds (Asclepias spp.), which result from only16 elongate cells that branch and spread through most above-ground tissues (Wilson 1986). Suchremarkably long and multinucleate branching laticifers are also known from cytological studies ofother species, such as Jatropha dioica (Euphorbiaceae), where 5 to 7 cells make up the entire laticifernetwork (Cass 1985). Articulating laticifers form loops and are often connected by perforationsin the cell walls of neighboring laticifers. Articulating laticifers, such as those produced in theAsteraceae and Caricaceae, are produced by larger chains of cells that form net-like structures,and tend to deliver latex much more comprehensively to local tissues.

All plant parts can contain latex. The commonly examined tissues of latex-bearing plants arestem and leaf tissue. Indeed, we are not aware of latex-producing plants that do not exude the latexin stems and leaves. Exudation of latex in roots appears more variable. Asclepias species apparentlydo not exude latex from roots, although at least a few species have laticifers in root tissues; otherspecies, such as those in the Asteraceae, exude copious latex from roots (Lucansky & Clough 1986;A.A. & K.K., personal observations). Latex exudation from reproductive tissues (buds, flowers,and fruits) is commonly observed, but like root latex, is far less studied than stem and leaf latex.Laticifers can transport latex and defensive substances upward of 70 cm to the damaged (exuding)points, as was demonstrated for the milkweed rubber vine Cryptostegia grandiflora (Buttery &Boatman 1976).

HISTORY AND HYPOTHESES

Like many plant defenses, latex has been observed, described, and used by humans for thousandsof years (Mahlberg 1993). Historically, latex was classified by its often opaque sticky exudation,and the propensity to coagulate upon exposure to air. As early at the 1600s, the term latex was used

www.annualreviews.org • Latex as Plant Defense 313

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a

b c

d e

Flow of latexduringexudation

Exuded latex accumulatesat the point ofdamage

Intact laticiferInactivated laticifer

Trench

Vein cut

Nonarticulated laticifer Articulated laticifer

f

Figure 1Latex exudes from the damaged points soon after the wounding by herbivores, and latex flow is caused byinternal pressure. (a) The latex exists throughout the laticifer before insect attack (dark green) andconcentrates at the point of damage (red ). (b) Vein-cutting behavior by specialists feeding on nonarticulatedlaticifers and (c) trenching behavior by specialists feeding on articulated laticifers. (d ) Vein-cut (arrow) madeon a leaf of Ficus virgata (Moraceae, a wild fig with nonarticulated laticifers) by the larvae of Cylestis thyodamas(Nymphalidae) (Ishigaki Island, Okinawa, Japan); (e) larvae of Erinnys alope (Sphingidae) trenching on Caricapapaya (Caricaceae, papaya with articulated laticifers) (photo courtesy of David Dussourd); and ( f ) a firstinstar monarch butterfly larva Danaus plexippus employing a circle trench on milkweed with nonarticulatedlaticifers (although monarchs typically use vein cutting when larger, first instar larvae employ trenching,presumably because of their small size).

by English-speaking physicians, its function was analogized to lymphatic vessels of animals, and itwas studied in several plant families (Mahlberg 1993). In a classic essay describing North Americanmilkweeds, James (1887) apparently proposed the first defensive hypothesis for latex, “. . .it carrieswith it at the same time such disagreeable properties that it becomes a better protection to the plantfrom enemies than all the thorns, prickles, or hairs that could be provided. In this plant, so copiousand so distasteful has the sap become that it serves a most important purpose in its economy.”


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Experimentally, only a few years after James’ essay, Kniep (1905) in Germany, published resultsof the first experiments to demonstrate latex as a resistance trait. He repeatedly damaged leavesof a plant in the Euphorbiaceae until latex no longer flowed from new cuts. Slugs readily ate suchleaves, but refused to eat the leaves of intact plants that were not drained of their latex. Nearly acentury later, Dussourd & Eisner (1987) showed that such disarming of the latex response, thatis, severing of the laticifers, was routine maintenance before meals in most of the mandibulateherbivores of milkweeds. An additional smoking gun implicating the adaptive role of latex comesfrom the observations that upward of 30% of newly hatched monarch butterfly caterpillars (Danausplexippus) die mired in latex of Asclepias humistrata (Zalucki & Brower 1992).

Other explanations for the production of latex have included functions involved in primarymetabolism, namely storage and movement of plant nutrients, waste, and maintenance of waterbalance. Essentially none of these functions remain credible and none have any empirical support(Farrell et al. 1991, Hunter 1994, Mahlberg 1993). For example, carbohydrates in Euphorbia latexare unavailable to the plant, even when light starved (Nissan & Foley 1986). Although the costsof latex production have not been estimated for any plant species (see section on Evolultion ofLatex), the fact that latex is often highly concentrated with secondary metabolites, carbohydrates,and enzymes suggests that it is not a waste product. In H. brasiliensis, latex can be up to 60% drymass rubber and 2% nitrogen (Shukla & Krishna Murti 1971); sugar-mimic alkaloids can make upto 18% of dry mass latex in mulberry, 100 times higher than the average concentration in leaves(Konno et al. 2006).

Studies of banana (Musa sp. Musaceae) indicate that the concentration of latex during exuda-tion is correlated with the turgor pressure of the plant, and Milburn and colleagues (Kallarackalet al. 1986, Milburn et al. 1990) have suggested that latex may be important in maintaining turgorvia osmoregulation. In particular, banana latex contains lutoids, which are transparent vesiclesthat compartmentalize various inclusions and have osmotic activity. Although a relationship be-tween leaf turgor and latex concentration has also been found for Neruim oleander (Apocynaceae)(Downton 1981), no studies have shown that specific attributes of the latex actually function tohelp maintain plant water balance. It is more likely that latex simply responds to turgor pressure.

EVIDENCE FOR LATEX AS A DEFENSE

Various forms of evidence, from the observational to the experimental and comparative, have beenaccumulated in support of latex as a plant defense against herbivory. Nonetheless, few studies todate have linked variation in plant production of latex to plant fitness (Agrawal 2005a). In thestrict sense, a plant defense is any trait that improves the fitness of plants in the presence (but notthe absence) of herbivores (Karban & Baldwin 1997). Thus, although we have assumed that latexis defensive (and hence adaptive for the plant), most studies have focused on latex as a trait thatreduces herbivory or the preference or performance of herbivores.

As a follow-up to the classic experiments of Kniep described above, Zalucki and colleagues haveconducted several studies demonstrating that depressurizing the latex of milkweeds (Asclepias spp.)increased the fitness (survival or growth) of specialized monarch butterfly caterpillars (Danausplexippus). By partially severing a leaf’s petiole, the flow of latex to that leaf can be essentiallystopped without altering the turgidity of the leaf. This treatment substantially improved fitness ofmonarchs on four milkweed species with high latex flow, but had relatively minimal effect on larvaewhen four milkweed species that produce less latex were treated (Zalucki et al. 2001b; Zalucki &Malcolm 1999). These experiments provide convincing evidence for a role of latex in resistanceto a specialist herbivore. Konno et al. (2004) further showed that washing papaya and fig leavesfree of their latex made them acceptable to herbivores that typically perish on intact leaves.


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Several studies have added latex to artificial diets or painted leaves with latex to assess its role inresistance. For example, latex of the milkweed Hoodia gordonii proved deterrent to larval feedingand adult oviposition by generalist cabbage loopers (Trichoplusia ni ) (Chow et al. 2005). Latexof a resistant variety of lettuce Lactuca sativa ‘Valmaine’ almost completely inhibited feeding ofDiabrotica balteata beetles when painted on leaves of a favored food (lima bean leaves) (Huang et al.2003). Nonetheless, latex from a different, susceptible variety of lettuce ‘Tall Guzmaine’ was notdeterrent (Sethi et al. 2008). This study implicated chemical compounds in the resistant latex (amoderately polar fraction of the latex) that were not present in the latex of the susceptible lettucevariety.

Quantitative genetic evidence demonstrates that latex is an important component of resistanceto herbivores. For example, in an analysis of 96 genetic lines of sweet potato (Ipomoea batatas), latexexudation was found to vary nearly 20-fold (Data et al. 1996). Application of this same latex toroot cores of sweet potato reduced feeding and oviposition by a specialist weevil, Cylas formicarius.Full-sibling genetic families of the common milkweed (Asclepias syriaca) can vary over fourfold intheir latex production when grown in a common garden (Agrawal 2005a, Van Zandt & Agrawal2004). This variation predicted growth of the specialist chrysomelid beetle Labidomera clivicollis(Van Zandt & Agrawal 2004), abundance of a cerambicid beetle Tetraopes tetraophthalmus (Agrawal2004), abundance and oviposition by a curculionid stem weevil Rhyssomatus lineaticollis (Agrawal& Van Zandt 2003), and overall community composition (Agrawal 2005a). Nonetheless, neithergenetic variation in latex within one species nor variation in latex across 24 milkweed species wasa good predictor of monarch caterpillar larval growth (Agrawal 2005a, Agrawal & Fishbein 2006).

Although the weight of evidence suggests that latex is defensive, and no strong alternativehypotheses have stood the test of time, the fitness benefits of latex production for plants have notbeen well quantified (see Agrawal 2005a and section on Evolution of Latex).

BIOCHEMISTRY AND MODE OF ACTION

Plant latex, exudates (including phloem sap), and resins contain various secondary metabolitesand proteins, often in concentrations that are much higher than in leaves. Indeed, the latex ofmost species contains a diversity of biologically active compounds. Many of these compoundsprovide resistance to herbivores via toxicity or antinutritive effects, whereas others are involved inthe stickiness that can mire insect herbivores. Several of these defense-related components (e.g.,rubber, cysteine protease, alkaloids, etc.) appear in latex of distant phylogenetic groups, suggestingcommon functions and convergent evolution. Below we survey the common constituents of latex,their mode of action, and possible biological effects on herbivores.

Secondary Metabolites

Rubber. Rubber (cis-1,4-polyisoprene) is a terpenoid found in the latex of many plant species,across some 300 genera and 8 plant families (Bushman et al. 2006, Metcalf 1967, Mooibroek &Cornish 2000). Both the stickiness and typically white color of latex are often derived from rubberparticles dispersed in the fluid. Rubber can make up a high concentration of fresh latex (e.g.,H. brasiliensis 44.3%, Ficus spp. (Moraceae) 15–30%, Alstonia boonei (Apocynaceae) 15.5%,Parthenium argentatum (Asteraceae) 8%) (Mooibroek & Cornish 2000). Given its high concentra-tions, uniform structure, and widespread distribution among unrelated plant families, rubber inlatex has likely convergently evolved, suggesting an important adaptive function. At present, it isgenerally accepted that the primary functional role of rubber in latex is to produce stickiness thatentraps whole insects (Dussourd 1993, 1995) or mires their mouthparts (Dussourd & Eisner 1987).


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Additionally, rubber is involved in sealing leaf wounds, which should prevent further drainage oflatex and may prevent infection by pathogens. However, the mechanisms of these effects (evenfor stickiness) are not well documented. We propose that the stickiness of latex may be causedby at least three factors: (a) elasticity of cis-polyisoprene, (b) coagulation of rubber particles, and(c) adhesiveness of rubber particles to the surfaces of insects.

Alkaloids. Alkaloids are basic (as opposed to acidic) natural products containing nitrogen, manyof which are toxic and typically do not have a primary function in plants. Alkaloids are producedby a variety of animals, microorganisms, and plants and have been reported from the latex ofmany species, sporadically distributed among angiosperm families, including Papaveraceae andMoraceae. For example, isoquinoline alkaloids such as chelidonine, sanguinarine, and copticinecan total nearly 20% fresh mass of the latex in Chelidonium majus (Tome & Colombo 1995).Sanguinarine affects neurotransmission by inhibiting choline acetyl transferase, various neurore-ceptors, and also DNA synthesis (making it toxic to insects and vertebrates alike) (Schmeller et al.1997). Morphine is produced in the latex of Papaver sominiferum (opium poppy) up to 5% freshmass latex (Itenov et al. 1999). It was recently found that synthetic enzymes involved in the earlystage of alkaloid synthesis are localized in parenchymal cells surrounding laticifer cells and thatthose involved in the late stages of synthesis were localized inside the laticifer (Samanani et al.2006, Weid et al. 2004).

Recently, sugar-mimic alkaloids were found in the latex of mulberry species (Morus spp.,Moraceae), and occurred up to 2.5% fresh mass (18% dry mass) in latex (Konno et al. 2006).Sugar-mimic alkaloids, also known as imino sugars, are potent inhibitors of various glycosidasesand sugar-metabolizing enzymes (Asano et al. 2000). These compounds show toxicity and growthretardation in insects by inhibiting sucrase in the midgut and trahalase in various other tissues,which results in the inability to uptake sucrose and utilize trehalose (Hirayama et al. 2007).

Cardenolides. Cardenolides, or cardiac glycosides, inhibit Na+/K+-ATPases, which are impor-tant for maintenance of electric potential in most animal cells, making them remarkably toxic to awide array of animals (Malcolm 1991). Latex of many plants in the Apocynaceae contains carde-nolides, and these range from trace amounts up to 30% dry mass of latex (Malcolm 1991, Seiberet al. 1982). Additionally, latex of Antiaris toxicaria (Moraceae) in tropical Southeast Asia containscardenolides (toxicariosides), which have been used as dart poisons (Carter et al. 1997). Cardeno-lides have also convergently evolved in a few other plant families (e.g., Brassicaceae, Celastraceae,Fabaceae), but in these cases they are not associated with latex (Malcolm 1991). In neuronal cells,cardenolides impact neurotransmission by binding to the amino acid asparagine at position 122 inthe extracellular region of Na+/K+-ATPase, which is present in most animals including humansand Drosophila (Holzinger et al. 1992, Holzinger & Wink 1996).

Most cardenolide-containing plant species produce a diversity of compounds, due in partic-ular to the different chemical structures of the glycosides in the molecule. Differences in thepolarity of cardenolides, in particular, have been linked to differential absorption in the animalbody, and thus with potentially differential toxicity. For example, nonpolar digitoxin is almostcompletely absorbed, irrespective of where it is administered to insects; conversely, ouabain, ahighly polar cardenolide, is intestinally absorbed quite slowly (Malcolm 1991). Nonetheless, theadaptive significance of cardenolide diversity is unknown. Virtually nothing is known about wherethe cardenolides are produced and how they are transported into the latex (Groeneveld 1999).Cardenolides have no known functions in plants other than defense.


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Terpenoids. Terpenoids (including rubber) are an extremely diverse group of carbon-based com-pounds that are derived from five-carbon isoprene units. Terpenoids likely have many functions inplants, including pollinator attraction, defense, and roles in primary metabolism (e.g., carotenoidsthat provide additional pigments for harvesting light energy), and can be produced abundantly inlatex. The latex of Lactuca sativa (cutivated lettuce) contains several sesquiterpene lactones (SL), in-cluding lactucin and the total SL concentration in the bolting stage of lettuce reached 147.1 mg/mllatex (Sessa et al. 2000). In particular, the concentration of lactucopicrin oxalate in latex wasover 1000 times higher in concentration than that in foliar tissue. In addition to those consti-tutive SLs, lettucenin A is induced in latex by fungal and bacterial challenge. Lactucopicrin and8-deoxylactucine are known to deter feeding by locusts (Rees & Harborne 1985), and lactucin trig-gers leaf-trenching by Trichoplusia ni, a facultatively trenching caterpillar (Dussourd 2003). TheseSLs have some antifungal activity against the pathogenic, Cladosporium herbarum; and lettuceninA, which is induced in latex by microorganisms, strongly inhibited the growth of Cladosporiunherbarum (Sessa et al. 2000).

The latex of some Euphorbiaceae, such as Euphorbia biglandulosa, contains diterpenes such asphorbol and its derivatives (Noack et al. 1980). These compounds have toxicity against insects andmammals, have tumor-promoting activity, and cause skin inflammation (Gershenzon & Croteau1991). Further, triterpenoids are reported as the major components of the latex of some Euphorbiaspp. (Mazoir et al. 2008).

Phenolics. Phenolics are a huge group of multifunctional carbon-based secondary metabolitesproduced by the shikimate pathway that include tannins, lignins, and flavonoids. Latex of thesweet potato, I. batatas (Convolvulaceae) contains high concentrations of hexadecyl, octadecyl,and eicosyl esters of p-coumaric acids (Snook 1994). The overall concentration of p-coumarateesters exceeded 3% fresh vine latex and 10% root latex of the variety “Jewel” (Snook 1994).The concentrations of (Z )-isomers of C16, C18, C20 coumarates inversely correlated with theacceptability by weevils, indicating that (Z )-coumarate esters may participate in the defense ofsweet potato against insect herbivores (Snook 1994). Additionally, the latex-like resin of Rhus spp.(lacquer plant, poison ivy, etc. in Anacardiaceae) are well known to contain urushiol, a catechol witha long carbon chain rich in double bonds and a compound known to cause strong skin irritations(Dawson 1954).

Proteins

Proteases. Proteases are enzymes that cleave protein and are found in all living organisms. Vari-ous types of proteases are found from latex of plants belonging to diverse phylogenetic clades. Forexample, cysteine proteases are reported from latex of plant families such as Caricaceae, Moraceae,Apocynaceae (Kimmel & Smith 1954; Arribere et al. 1998, Sgarbieri et al. 1964), and serine pro-tease from Moraceae, Euphorbiaceae, Apocybnaceae, Convolvulaceae (Arima et al. 2000, Tomaret al. 2008). The latex-like resin exudates of mango, Mangifera indica (Anacardiaceae), containboth serine and cysteine proteases (Saby et al. 2003). In spite of the abundance and frequent oc-currence of proteases in plants, an adaptive role of these compounds for plants was not suggesteduntil recently. Direct evidence for the involvement of cysteine proteases in plant resistance againstherbivores came from experiments showing that the strong toxicity of papaya and wild fig (Ficusvirgata) leaves against the Eri silkworm, Samia ricini, and the cabbage worm, Mamestra brassicae,disappeared when latex was washed out of the leaves or when E-64, a cysteine protease-specificinhibitor, was painted on the surface of leaves (Konno et al. 2004) (Figure 2). These resultsdemonstrated that cysteine proteases in papaya (papain) and fig (ficin) latex are a crucial part of


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Control papayaleaf with latex

Papaya withE–64

Washed papayaleaf strips

Castor oilplant leaf

2 cm

Figure 2Defensive activity of papain, a cysteine protease from latex of Carica papaya. (a) Second instar Eri silkworm,Samia ricini, fed intact (clipped from the petiole) leaves containing latex died; (b) leaf toxicity was lost afterpainting E-64, a cysteine protease-specific inhibitor, on the leaf surface, or (c) removing the latex by washingit out of leaf strips; (d ) larvae on these treated leaves grew as well as others that were fed their natural hostplant, Ricinus communis (modified from Konno et al. 2004). The photo was taken on day 4.

plant resistance (Figure 2). The protease activity in papaya latex is 20 times higher than the lethalconcentration of papain to nonadapted herbivores, and nearly 200 times higher in concentrationin latex compared to that in leaves (Konno et al. 2004).

Proteases are digestive (that is, they break apart protein) and are commonly found in animalguts. Thus, their role as a plant defense appears to be a remarkable turn on the plant-insectinteractions, essentially plants eating the insect! Nonetheless, the mechanisms of protease toxi-city have not well studied except for Mir1-CP, which is toxic to insects and accumulates at thesite of larval feeding in maize lines resistant to caterpillars (Pechan et al. 2000). Here, the cys-teine proteases degrade the peritrophic membrane of the insect midgut, which consists of pro-teins and chitin (Pechan et al. 2002). The observation that the dead bodies of caterpillars miredin latex of papaya, fig, and milkweed turn black and soft (A.A. Agrawal & K. Konno, unpub-lished data) indicates that all tissues of insects are a potential target of digestion by proteases inlatex.

Protease inhibitors (PIs). PIs are thought to function as antinutritive secondary metabolites bybinding to proteases and preventing the digestion of protein. Trypsin (serine protease) inhibitorsare found in latex of Ficus carica (Kim et al. 2003) and Carica papaya (Azarkan et al. 2004). Geneexpression of trypsin inhibitors is also in the laticifers of H. brasiliensis (Han et al. 2000). Further-more, the latex-like ploem sap of Cucurbita maxima (pumpkin, Cucurbitaceae), contains varioustypes of protease inhibitors including trypsin, chymotrymsin, and cysteine or aspartic inhibitors(Kehr 2006, Walz et al. 2004). Protease inhibitors inhibit proteolysis and utilization of proteins,and their defensive roles against herbivores and fungi are well-established in many plants withoutlatex (Ryan 1990, Zhu-Salzman et al. 2008). Trypsin inhibitor, a class-II chitinase and a glutaminylcyclase, is absent from latex of undamaged leaves, but was strongly induced in latex after damage(Azarkan et al. 2004).


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Lectins and hevein-like chitin-binding proteins. Lectins are carbohydrate-binding proteinsthat have affinity with specific sugar moieties, which often have toxic activities against animalsincluding insects (Van Damme et al. 1998). Several types of lectins have been found in latexfrom Euphorbiaceae, Moraceae, Apocynaceae, and phloem sap from Cucurbitaceae. Of these,hevein, the major latex protein from H. brasiliensis is important in the agglutination of rubberparticles (Gidrol et al. 1994), and its m-RNA is induced by wounding (Broekaert et al. 1990).Upon exposure to air, hevein binds to receptor proteins and cross-linked rubber particles, therebycausing coagulation of latex.

The mechanisms of coagulation have also been studied in the latex-like phloem sap of cucurbits(Read & Northcote 1983, Kehr 2006). After exudation, two major proteins in the sap, PPI andPPII, coagulate and form a fibrous structure (both proteins are soluble in the intact phloem).When in contact with air, PPI and PPII molecules are crosslinked through S-S bonds, formedamong cysteine residues. Coagulation of cucurbit phloem sap not only stops exudation but alsoglues mouth parts of beetles and can inhibit feeding (McCloud et al. 1995).

The chitin-binding MLX56 proteins from Morus latex are highly toxic to many caterpillarsincluding the cabbage worm, Mamestra brassicae (Wasano et al. 2009). Proteins (including chitin-binding and chitinolytic proteins and cysteine proteases) extracted from the latex of the milkweedCalotropis procera were shown to have insecticidal action against four different crop pests whenincorporated into artificial diets (Ramos et al. 2007). Chitin-binding proteins with hevein-likedomains, such as the wheat germ lectin, are toxic and inhibit the synthesis of the insect gutperitrophic membrane (Hopkins & Harper 2001, Van Damme et al. 1998).

Chitinases. Chitinases, enzymes that degrade chitin (important components of insects’ gutperitrophic membrane), are widely found in plant latex from several plant families includingCaricaceae, Moraceae, and Euphorbiaceae (Howard and Glazer 1969; Glazer et al. 1969; Lynn& Clevette-Radford 1987). Because chitin is the major constituent of the cell wall of fungi, it isreasonable to assume that chitinases protect the leaves from infection by pathogenic fungi as well.Expression of chitinases in the latex of F. carica and C. papaya increases in response to woundingor treatment with jasmonic acid (the plant hormone involved in signal transduction of plant re-sponses to herbivory) (Azarkan et al. 2004, Kim et al. 2003). Chitinases from insect origins showtoxic effects on other insects when orally ingested (Kramer & Muthukrishnan 1997, Kabir et al.2006), suggesting that chitinases in latex may have a defensive role. Nonetheless, the toxic effectsand defensive roles of plant chitinases are not well established, except for chitinases of poplar trees(not in latex), which are induced in response to herbivory and provide protection again subsequentattack (Lawrence & Novak 2006).

Oxidases. Polyphenol oxidase (PPO) and peroxidase (POD) are common plant oxidases reportedfrom Euphorbiaceae, Moraceae, and Anacardiaceae (Saby et al. 2003, Wititsuwannakul et al.2002). PPOs and some PODs are regarded as plant antiherbivore defense proteins, because theyoxidize mono- or di-hydroxyphenolics that are ultimately converted in o-quinones, which thencovalently bind to amino acids such as cysteine and lysine, making them inaccessible, and decreasethe nutritive value of leaf protein (Felton et al. 1992, Zhu-Salzman et al. 2008). The frequentbrowning of latex upon exposure to air in many plant species suggests that PPOs and PODs maybe widely distributed in latex. Proteomic analyses of phloem sap exuded from cucumber, Cucurbitasativa and pumpkin, Cucurbita maxima, revealed abundant lipoxygenases (LOX) (Walz et al. 2004).LOXs are implicated as defense proteins since they are often induced by wounding or jasmonicacid, and since hydroperoxides formed by the oxidation of linolenic/linolenic acids by LOXs may


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Trenching or vein-cutting: the behaviorof deactivatingpressurized laticifersby insects, after whichthey typically feed ondistal tissues free oflatex exudation

react with amino acids, in addition to the loss of fatty acids essential for insects (Felon et al. 1994,Zhu-Salzman et al. 2008).

Others. In addition to the above-described proteins that were reported from many plant groups,some latex proteins are confined to specific plant taxa and have been suggested to be in-volved in plant defense. These compounds include phosphatase in Euphorbiaceae (Lynn &Clevette-Radford 1987); lipase in Caricaceae, Euphorbiaceae, Apocynaceae (Fiorillo et al. 2007,Gandhi & Mukherjee 2000, Giordani et al. 1991); glutaminyl cyclase in Caricaceae (papaya)(Azarkan et al. 2004, Zerhouni et al. 1998); and gum arabic glycoprotein, a high-molecular-weight,hydroxyproline-rich arabinogalactan-protein found from exudates of Acacia senegal (Fabaceae)(Goodrum et al. 2000). Finally, linamarase in cassava leaves and latex is a β-glucosidase thatspecifically degrades linamarine, also present in the leaves and roots of the same plant, and resultsin the production of hydrogen cyanide that is toxic to most organisms. The linamarase activityreported from the latex of cassava was more than 300-fold higher than that in its leaves (Nambisan1999).

HERBIVORE ADAPTATIONS FOR FEEDINGON LATEX-BEARING PLANTS

Many herbivorous insects, particularly specialists, have adaptations to cope with the toxins andantinutritive agents in the leaves (and latex) of plants. For example, larvae of monarch butterfliesspecialized in feeding on milkweeds exuding cardenolide-containing latex have evolved Na+/K+-ATPases insensitive to cardenolides (Holzinger & Wink 1996), and this ability was convergentlyevolved in other insect groups such as Chrysochus beetles feeding on Apocynum (Labeyrie & Dobler2004). The silkworm, Bombyx mori, a specialist feeding on mulberries (Morus spp.) exuding la-tex containing sugar-mimic alkaloids, has evolved sucrase and trehalase insensitive to sugar-mimicalkaloids (Daimon et al. 2008, Hirayama et al. 2007). Below we focus on behavioral tactics that her-bivores employ to reduce exposure to both the sticky physical aspect of latex and the concentratedphytochemicals in latex.

Many insects have evolved behaviors to inactivate the pressurized delivery system of lati-cifers. Since the laticifer system relies upon the ability to transport defense substances (Figure 1),their functions are lost when the transport routes are disrupted (Dussourd 1993, 1999;Dussourd & Denno 1991, 1994; Dussourd & Eisner 1987). The loss of function is especially pro-nounced in nonarticulated laticifers (branching type) without bypassing loops (Figure 1). Cuttingof the laticifers at a single location upstream of feeding can deactivate all downstream activity(Figure 1). Many mandibulate herbivores of latex-bearing plants with nonarticulated laticifers ac-cordingly engage in vein-cutting behavior (Figure 1) (Dussourd 1993, Dussourd & Denno 1991,Dussourd & Eisner 1987). Plants with articulated laticifers (net or web type) (Figure 1) are betterprotected from herbivory because, even when insects cut veins, there are circuitous routes for latexto go downstream of the cut. Specialists feeding on leaves with articulated laticifers typically showa behavior called trenching (Figure 1), in which insects cut a leaf-wide trench or circle trench(Figure 1) (Dussourd 1993; Dussourd & Denno 1991, 1994). Whether herbivores employ vein-cutting or trenching corresponds well to the types of laticifer (that is, nonarticulate or articulated)of their host plants (Dussourd & Denno 1991).

The effects of these behaviors are quite evident from experiments showing that natural orartificial trenching or vein-cutting on latex-exuding leaves can reduce the amount of exuding latexand render leaves edible to herbivores not typically adapted to these plants (Dussourd & Denno1991, 1994; Dussourd & Eisner 1987, Zalucki & Brower 1992). Just as latex is a widely-distributed


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trait and is likely to have evolved independently multiple times, so too are trenching and vein-cutting behaviors. For example, vein-cutting is observed in Orthoptera (Tettigoniidae), Coleoptera(Cerambycidae, Chrysomelidae, Curculionidae), and Lepidptera (Arctiidae, Gelechiidae,Noctuidae, Nymphalidae, Pyralidae), and trenching is observed in Coleoptera (Coccinellidae,Chrysomelidae) and Lepidoptera (Noctuidae, Nymphalidae, Sphingidae) (Dussourd & Denno1991; A.A. Agrawal & K. Konno, personal observations). Such convergence is evident even onsingle host plants. Indeed, on common milkweed (Asclepias syriaca) in eastern North America,vein-cutting is commonly exercised by Arctiidae, Cerambycidae, Chrysomelidae, Curculionidae,and Nymphalidae (Dussourd & Eisner 1987, Fordyce & Malcolm 2000).

Not all herbivores on laticiferous plants trench or cut veins. For example, adult Chrysochusauratus (Chrysomelidae) feed on latex-rich Apocynum spp. and show population variation inwhether or not they cut veins (Williams 1991; A. Agrawal, personal observations). Other her-bivores, such as the milkweed leaf miner (Liriomyza asclepiadis Diptera), apparently feed withoutcoming into contact with latex (Agrawal 2005a). Most sap suckers (Hemiptera) similarly do notcontact latex because of their intercellular feeding, and thus have no obvious adaptations forfeeding on latex-bearing species. Asclepias syriaca is host to at least five hemipterans (three aphidsand two lygaeid bugs) (Agrawal 2005a, Smith et al. 2008; A.A. Agrawal, personal observations).Nonetheless, latex can occasionally entrap and kill hemipteran sap feeders, as was demonstratedfor aphids and whiteflies on lettuce (Dussourd 1995).

Trenching and vein-cutting is a phenotypically plastic behavior. Many species will not engagein the behavior if feeding on an already depressurized leaf. It has been recently found that trench-ing and vein-cutting behavior in insects is also specifically triggered by compounds in latex andexudates (Dussourd 1997, 2003; Helmus & Dussourd 2005). The cabbage looper, Trichoplusia ni,cuts trenches on plants that produce exudates such as Lactuca sativa (latex), parsley (Petroselinumcrispum, Apiaceae, oil from oil ducts), cucumber (Cucumis sativus, Cucurbitaceae, exudates fromphloem), and cardinal flower (Lobelia cardinalis, Campanulaceae, latex), but it does not trench onplantain (Plantago lanceolata, Plantaginaceae), which does not have an exudate. When exudatesfrom the above exuding species were applied orally to the cabbage looper beforehand, the looperstrenched on plantain leaves (Dussourd 1997, 2003). The triggering stimulants include lactucin(a sesquiterpene lactone) from lettuce latex, myristicin (a phenylpropanoid) from parsley oil, andlobeline (an alkaloid) from cardinal flower. Each of these compounds is known or suspected to betoxic; in other words, a diverse set of toxins are the cues used to induce trenching. The trenchingstimulants from cucumber phloem exudates, however, are still unknown, but are certainly not cu-curbitacins (Dussourd 2003, McCloud et al. 1995). Similarly, toxic furanocoumarins from parsleydo not trigger trenching (Dussourd 2003). Finally, monarch caterpillars, specialists of milkweeds,do not initiate vein-cutting in response to cardenolides; some other nonproteinaceous and non-adhesive fraction of milkweed latex induces trenching, although the specific compounds have notbeen identified (Helmus & Dussourd 2005).

PHENOTYPIC PLASTICITY OF LATEX PRODUCTION

Several lines of evidence suggest that latex production in plants is phenotypically plastic (thatis, responsive to environmental conditions). For example, work on the rubber tree H. brasiliensisand sweet potato (I. batatas Convolvulaceae) shows that light levels, drought, and soil moistureconditions determine the amount of latex production (Data et al. 1996, Raj et al. 2005). In a recentstudy of several Asclepias species, we have shown that soil moisture, soil fertility, and leaf herbivorycan all affect latex production (Figure 3). Several important results emerge from these data. First,closely related species vary nearly tenfold in their constitutive latex production. Second, all three


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Late

x (m

gs)

0

1

2

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4ControlLow soil moistureHerbivoryFertilization

A. angusti

folia

A. boliv

iensis

A. cura

ssavica

A. fasc

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A. incarn

ata

A. pum

ila

A. subverti

cillata

A. texana

Figure 3Impacts of environmental conditions (soil moisture, leaf herbivory by monarch caterpillars, and fertilization)on the production of latex (mean ± SE) in eight milkweed species. Latex was measured on plants grownfrom seed in a common growth chamber environment by clipping the tip of a leaf and measuring the freshmass of exuded latex. These eight species are close relatives, all in the Series Incarnatae of the genus Asclepias.Data from S.C. Cook, A.C. Erwin, M. Fishbein & A.A. Agrawal, unpublished manuscript.

environmental factors examined had considerable effects on latex production, but the influencewas reversed in different species. For example, although herbivory reduced latex production inA. angustifolia, it substantially induced latex production in several other species. Third, the rankorder of species for their latex production can be altered by environmental conditions. Thus, boththe biotic and abiotic environment can have a strong influence on latex exudation.

Despite the fact that essentially nothing is known about the adaptive value of phenotypicplasticity in latex production, the variation observed in Figure 3 is suggestive. For example,the nearly threefold increase in latex exudation following monarch herbivory in A. fasciculariscompared to no indication of induction in several other species (including those with equivalentconstitutive latex production) is suggestive of some adaptive function. In more recent work we havedemonstrated that jasmonic acid application is sufficient as an inducer of latex in some Asclepiasspecies (e.g., A. fascicularis) (S. Rasmann & A.A. Agrawal, unpublished data). This indicates thatthe near-universal plant hormone, jasmonic acid, which is responsible for the induction of sundrychemical defenses, trichomes, and extrafloral nectar throughout the angiosperms (Agrawal 2005b),is sufficient by itself to elicit an increase in latex exudation. In the rubber tree H. brasiliensis,jasmonic acid application increases the number of laticifers, and hence latex flow, in saplings(Hao & Wu 2000). Finally, there is some evidence for specificity in the induced latex response ofA. syriaca. We reported that herbivory by specialist leaf beetles (Labidomera clivicollis) resulted ingreater induction of latex compared to similar levels of herbivory imposed by monarch caterpillars(Van Zandt & Agrawal 2004). In addition to phenotypic plasticity in the amounts of latex beingexuded following damage, the many concentrated secondary plant compounds in latex may alsobe plastically expressed (see section on Biochemistry and Mode of Action).

There is additionally an ontogenetic and qualitative component to plasticity in latex exudation.Young sweet potato vines (I. batatas) produced up to fourfold more latex and had less weevilfeeding damage than older more mature portions of the vine (Data et al. 1996). A slightly differentpattern has recently been observed for common milkweed (A. syriaca), with young leaves showing


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equal levels of constitutive latex exudation as that of older leaves, but only the former showinginducibility following herbivory (R. Bingham & A.A. Agrawal, unpublished data). In addition tophenotypic plasticity in the quantity of latex exudation, the quality of latex is also plastic. Forexample, several defense-related proteins potentially involved in plant defense, such as chitinase,protease inhibitors, and glutaminyl cyclase, were induced in latex by damage of papaya leaves(Azarkan et al. 2004).

EVOLUTION OF LATEX

From a microevolutionary perspective, the Darwinian criteria necessary for a trait to evolve bynatural selection are that it must exhibit heritable variation that influences plant fitness. The onlydata available on heritability and selection (that is, differential fitness for varying phenotypes) onlatex come from a field common garden study of A. syriaca (Agrawal 2005a). Over two years, theheritability was estimated in 23 full-sibling families, and was 0.161 in the first year, and 0.368 inthe second year. In the same set of plants, latex exudation more than doubled in the second year ofgrowth compared with that in first-year seedlings, indicating a strong ontogenetic component tolatex production. In the second year of growth, genetic families from a single population showedover fivefold variation (between 0.8–4.5 mg latex exuded upon leaf damage). The relatively low, butstill statistically significant, heritable variation is consistent with a strong environmental influenceon latex production discussed above.

The genetic correlation between latex exudation and plant fitness from the study of A. syriacarevealed marginal evidence for directional natural selection to increase latex production (selectiongradient β = 0.285, P = 0.067) (Agrawal 2005a). Given that this study was conducted in the fieldwith ambient levels of herbivory, we conclude that any costs of latex were apparently outweighed bybenefits associated with reduced herbivory. No other estimates of heritability, costs, or benefits oflatex production for plants exist to our knowledge. The relative ease with which latex exudation canbe quantified makes it an exceptionally attractive system for such studies. One potential influenceon the adaptive evolution of latex is genetic correlations with other traits that affect fitness. ForA. syriaca, however, latex was not genetically correlated with plant production of foliar cardenolides,trichomes, toughness, or nitrogen content (Agrawal 2005a).

Latex production has been implicated as a key innovation that has spurred adaptive radiationin plants. Across the angiosperms, Farrell et al. (1991) showed that latex-bearing plant clades weresignificantly more species rich than were sister clades lacking latex (13 of 16 pairs showed thispattern). Across more closely related species, the dynamics of trait evolution can be inferred in aphylogenetic context. In some groups such as the Asteraceae, Apocynaceae, Euphobriaceae, andSapotaceae, many if not most species produce latex, and this is likely to be the ancestral conditionand to be evolutionarily conserved. Conversely, in other plant families (e.g., Aceraceae, Fabaceae,Salicaceae), latex may be derived and evolved in isolated taxa. Few systematic studies have examinedthe evolution of latex within clades.

Within Asclepias species, we have shown a pattern of phylogenetic decline in latex produc-tion as the genus diversified (Agrawal & Fishbein 2008, Agrawal et al. 2008). This pattern couldbe interpreted in two ways. First, reduced latex production is apparently associated with an in-creased diversification rate (within Asclepias). Alternatively, there could simply be evolution towardphenotypic declines in latex production within the group, and this may have little to do with diver-sification rate per se. It has been hypothesized that there may be selection for a macroevolutionaryreduction of defense traits when plants are associated with a herbivore fauna dominated by spe-cialists that can inactivate resistance mechanisms such as latex (Agrawal & Fishbein 2008, Agrawalet al. 2008).


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Trait correlations in comparative analyses that are independent of phylogenetic history re-veal either a strong physiological constraint that is unbreakable, even over macroevolutionarytimescales, or that the relationship is adaptive and has been maintained by natural selection. Suchtrait correlations have been employed in the analyses of plant defense syndromes, defined as theconvergent evolution of suites of correlated defense traits associated with some aspect of plantor herbivore ecology (Agrawal & Fishbein 2006). For latex in milkweeds, latex amount, cysteineprotease activity and trichome density show positive correlated evolution (Agrawal & Fishbein2006, 2008). Trade-offs were not evident between latex and several other defense traits, althoughwe did find some evidence for a trade-off with cardenolides in latex (but not in leaf tissue) (Agrawal& Fishbein 2006, 2008). Despite the correlated structure of plant defense evolution, it is possibleto find related milkweeds that are high in one defense trait and remarkably low in others (Zalucki& Malcolm 1999). For the resin canals of Bursera, Becerra et al. (2001) have reported similar cor-related evolution, with species that have highly pressurized exudation of terpenes (that is, squirtgun defense) tending to have highly volatile terpene mixtures; the terpenes of low-flow Burseraspecies are more complex, viscous, and less volatile.

SUMMARY POINTS: LATEX AS A DEFENSE SYSTEMBelow we summarize our view of how latex works as an effective, coordinated, and econom-ical defense system.

1. The average concentration of defense substance in latex is often much higher than thatfound in leaves (and measures of leaf chemistry often include residual contents of lati-cifers) (e.g., 50–1000 times higher concentrations of defense chemicals or proteins areobserved in latex of milkweeds, lettuce, and papaya; Seiber et al. 1982, Sessa et al. 2000,Konno et al. 2004), and this is what herbivores typically encounter when trying to con-sume plants with latex. Some compounds “stored” in latex may be autotoxic to the plant.Remarkably diverse defensive chemicals, including toxins and antinutritive compoundsfrom all major classes of secondary metabolites, and various types of defense proteins arefound in latex (see section on Biochemistry and Mode of Action).

2. Latex is mobilized and transported to the site of damage immediately after the damagee.g., latex can travel more than 70 cm to the damaged points in Cryptostegia grandiflora(Buttery & Boatman 1976). Since the defense substance accumulates at the point of dam-age (Figure 1a), latex is similar to an inducible defense system, although it is preformedin the plant.

3. Points 1 and 2 above indicate that latex is an economical system to provide a concentrateddefense when and where it is needed most, with relatively small total amount of defensesubstances deployed. Other plant defenses appear to be far less mobile, especially directlyfollowing damage. This economy may be indicative of why latex is advantageous andsuccessful as a defense against herbivory, and is consistent with its highly convergentevolution and apparent role as a key innovation in the radiation of plants.

4. Given points 1 and 2 above, latex can be seen as primarily effective against tiny mandibu-late insects (e.g., mortality of specialist caterpillars feeding on milkweeds are the highestin the earlier instars and especially high at the first bites after hatching (Zalucki et al.2001a,b). Larger herbivores that feed on whole plants (or even entire leaves) shouldbe much less affected because the accumulation of latex at the site of damage will beineffective and the exposure to defensive chemistry will not be concentrated.


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5. Because laticifers are only effective when they can transport defense substances underpressure, their function is easily lost when the canals are disrupted. Indeed, this is themost common method of counterdefense (that is, vein-cutting and trenching; Figure 1)and consumption by adapted herbivores. This strategy is highly convergent and hasevolved in three orders of insects.

6. The clotting activity of latex is essential so that defenses are not lost or wasted, wounds aresealed, and plant pressure is maintained. In most latex, clotting occurs in a few minutesafter the damage and exposure to air. Additionally, clotting of latex is involved in thestickiness that gums up insect mouthparts or whole bodies.

FUTURE ISSUESAlthough alternative hypotheses for nondefensive functions of latex have been discounted,there are still large gaps in our understanding. The ease with which latex exudation canbe estimated, the rich chemistry of compounds within latex, and the spectacular diversityand natural history of these plant-insect interactions make them ideal to study the ecology,evolution, and mechanisms of plant defense. For the future, we propose resolution of thefollowing outstanding questions:

1. What is the genetic basis of latex production? Although quantitative genetic variation inlatex production has been reported for a couple of systems, we know essentially nothingabout heritability, costs and benefits of latex, the number of genes involved, or the identityand molecular evolution of latex genes.

2. Given the diversity of phytochemicals and proteins in the latex of any given species, whatare the redundant, complementary, or synergistic effects of these components? What isthe level of specificity in elicitation and impact of latex on different guilds of herbivores?

3. Beyond the temperate-tropical comparison, in what ecological contexts is latex found?A comparative assessment of latex either within clades or communities would be helpfulto address this question.

4. Does latex play any role in tritrophic interactions (e.g., does time spent vein-cutting ortrenching expose herbivores to predators)? Are predators/parasitoids attracted to siteswhere fresh latex is exuding?

5. Are there novel antiherbivore defense chemicals or proteins in plant latex belonging tonew classes or with novel modes of action? Can these be exploited in plant breeding orpest management?

6. In addition to defense against herbivores, latex likely plays an equally important role inprotecting plants against microbial pathogens. What are the physical effects of latex onwound healing; how can microbial entrance to the plant be prevented; and what are thechemical effects of antimicrobial compounds in latex?

7. When did latex evolve in different plant lineages, and likewise, when did vein-cuttingand trenching evolve? Time-calibrated phylogenies should begin to allow us to answerthese questions, and may also address the original functions of latex.


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8. What, if any, adaptive role does latex play in species where it is exuded in only traceamounts (e.g., Acer platanoides and Asclepias tuberosa)?

DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings thatmight be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

A.A.A. and K.K. contributed equally to this review. We thank David Dussourd, Amy Hastings, IanKaplan, Sergio Rasmann, Sharon Strauss, Jennifer Thaler, and Meron Zalucki for discussions orcomments on the manuscript. This research was supported by NSF-DEB 0447550 to A.A.A. andby a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science(JSPS 20248007) and by a Research Grant Project from the Ministry of Agriculture, Forestry andFishery of Japan (MAFF) to K.K.

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agrawal and konno 2009 latex annual review

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